Two spaced bouts of moderate-intensity exercise reduce counterregulatory responses to a next-day hypoglycemic challenge and lead to hypoglycemia, a phenomenon that was characterized as exercise- associated autonomic failure (EAAF) and seen as detrimental to type 1 diabetics. We have recently found that a 20% lowering of blood glucose and altered pancreatic hormone response depend on exercise being performed in fasted/postabsorptive state, as none of the changes occurs when exercise is carried out in post- prandial state. We hypothesize that inadequate hepatic glycogen recovery due to preferential muscle glucose uptake during and after exercise is responsible for EAAF and is mediated by increased secretion of insulinotropic gut peptides glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP), that increase insulin/glucagon ratio and reduce glucoregulatory setpoint. The aims of this study are to (1) document the magnitude of increase in muscle glucose uptake and reduction in HGO in response to two spaced bouts of exercise in fasted state compared to the same exercise carried out after meals and in sedentary state;(2) characterize the neuroendocrine changes responsible for lowering of plasma glucose after spaced exercise in fasted state, (3) determine whether a 50% reduction in exercise dose in fasted state coupled with a 50% reduction in dietary carbohydrate intake is as effective in lowering blood glucose as the longer exercise and higher dietary carbohydrate content, and (4) manipulate hepatic glucose availability to determine whether hepatic glycogen repletion rather than the exercise protocol is responsible for sustained lowering of plasma glucose. We will assess muscle glucose uptake and HGO through measurements of stable glucose isotope with mass spectrometer (aim 1);measure changes in plasma concentrations of GLP-1, GIP, insulin, and counterregulatory hormones (aim 2) during manipulations described under aims (1);assess the effectiveness of a scaled-down exercise dose and carbohydrate meal content by 50% (aim 3);and lower and increase the level of hepatic glycogen repletion by, respectively, limiting evening dietary carbohydrates to 20 g, or providing a high carbohydrate evening meal and overnight intravenous glucose infusion that will match nocturnal glucose utilization (aim 4). This study will analyze the neuroendocrine mechanism of EAAF and provide an effective and manageable exercise and dietary prescription for prevention of type 2 diabetes. PUBLIC HEALTH RELEVANCE: The timing of two daily bouts of exercise before the meals lowers blood glucose concentration. This study examines the role of liver glycogen depletion in this phenomenon and seeks to define the timing of meals and exercise and dietary carbohydrate content that are necessary to produce it.